Abstract P1073: Pathways Regulating Cardiac Myocyte Proliferation

Introduction: Several pathways have been shown to promote cardiomyocytes (CMs) proliferation and regeneration including the Hippo signaling pathway. Our lab demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases the proliferation of adult CMs (ACMs). However, all these stimulations are insufficient to promote robust ACM proliferation. The effect of combining these interventions on CM proliferation and myocardial regeneration remains unknown. Hypothesis: KDM4D-mediated CM-specific H3K9 demethylation and Hippo pathway inhibition have additive roles in promoting CM proliferation. Methods and results: miR-199 overexpression preferentially increased S-phase CMs (EdU + ) in cultured neonatal rat ventricular cardiomyocytes (NRVMs), while H3K9me3 demethylase KDM4D preferentially increased G2/M markers (phospho-H3). The combination of KDM4D and miR-199 increased total cell number of NRVMs compared to either alone. Since the effects of miR-199 on CM proliferation are mediated through inhibition of Hippo signaling, we inhibited the pathway directly via knock-down of Salvador Family WW Domain Containing Protein 1 (Sav1). Similarly, Sav1 inhibition also led to S-phase reactivation and additional cell cycle re-entry was seen when combined with KDM4D overexpression. Inducibly activating KDM4D (iKDM4D) in adult transgenic mice together with shRNA-mediated knock-down of Sav1 (iKDM4D+Sav1-sh) resulted in a significant increase in cycling CMs compared to either intervention alone. In vitro gene expression analysis demonstrated that KDM4D preferentially induced expression of genes regulating late (G2/M) phases of the cell cycle while miR-199 and si-Sav1 preferentially upregulated genes involved in G1/S phase. RNA-seq and promoter analysis demonstrated that KDM4D and Hippo signaling pathway regulate CMs proliferation through different transcriptional pathways. KDM4D upregulated E2F1 and FoxM1 expression, whereas miR-199 and si-Sav1 induced Myc. Conclusions: KDM4D additively induces CMs proliferation with the Hippo-Yap1 pathways. This may have important implications for strategies that target cardiac regeneration in treating heart disease.